Polyketides and nonribosomal peptides are two large families of natural products that include many clinically valuable drugs, such as erythromycin and vancomycin (antibacterial), FK506 and cyclosporin (immunosuppresant), and epothilone and bleomycin (BLM) (antitumor). The biosyntheses of polyketides and nonribosomal peptides are catalyzed by polyketide synthases (PKSs) (Hopwood (1997) Chem. Rev. 97: 2465; Katz (1997) Chem. Rev., 97: 2557; C. Khosla, (1997) Chem. Rev., 97: 2577; Ikeda and Omura, (1997) Chem. Rev., 97: 2591; Staunton and Wilkinson (1997) Chem. Rev., 97: 2611; Cane et al. (1998) Science 282: 63) and nonribosomal peptide synthetases (NRPSs) (Cane et al. (1998) Science 282: 63. Marahiel et al. (1997) Chem. Rev. 97: 2651; von Döhren et al. (1997) Chem. Rev. 97: 2675), respectively. Remarkably, PKSs and NRPSs use a very similar strategy for the assembly of these two distinct classes of natural products by sequential condensation of short carboxylic acids and amino acids, respectively, and utilize the same 4′-phosphopantetheine prosthetic group, via a thioester linkage, to channel the growing polyketide or peptide intermediate during the elongation processes.
Both type I PKSs and NRPSs are multifunctional proteins that are organized into modules. (A module is defined as a set of distinctive domains that encode all the enzyme activities necessary for one cycle of polyketide or peptide chain elongation and associated modifications.) The number and order of modules and the type of domains within a module on each PKS or NRPS protein determine the structural variations of the resulting polyketide and peptide products by dictating the number, order, choice of the carboxylic acid or amino acid to be incorporated, and the modifications associated with a particular cycle of elongation. These features of PKS and NRPS inspired us to search for a hybrid PKS and NRPS system. Since the modular architecture of both PKS (Cane et al. (1998) Science 282: 63; Katz and Danadio (1993) Ann. Rev. Microbiol. 47: 875 (1993); Hutchinson and Fujii (1995) Ann. Rev. Microbiol. 49: 201) and NRPS (Cane et al. (1998) Science 282: 63, Stachelhaus et al. (1995) Science 269: 69; Stachelhaus et al. (198) Mol. Gen. Genet. 257: 308; Belshaw et al. (1999) Science 284, 486) has been exploited successfully in combinatorial biosynthesis of diverse “unnatural” natural products, it is imagined that a hybrid PKS and NRPS system, capable of incorporating both carboxylic acids and amino acids into the final products, could surely lead to even greater chemical structural diversity.
The BLMs, differing structurally at the C-terminal amines of the glycopeptides, are a family of antibiotics produced by Streptomyces verticillus (Sv). BLMs exhibit strong antitumor activity through a metal-dependent oxidative cleavage of DNA or RNA in the presence of molecular oxygen and are incorporated into current chemotherapy of several malignancies under the trade name of Blenoxane® that contains BLM A2 and BLM B2 as the principal constituents (Sikic et al. Eds. (1985) Bleomycin Chemotherapy, Academic Press, New York; Natrajan and Hecht (1994) pages 197-242 In: Molecular Aspects of Anticancer Drug-DNA Interaction Vol. 2, Neidle and Waring Eds., Macmillan, London). Umezawa, Fujii, Takita, and co-workers extensively studied the biosynthesis of BLM in Sv ATCC15003 by feeding isotope-labeled precursors and by isolating various biosynthetic intermediates and shunt metabolites, establishing that the BLMs are in fact natural hybrid metabolites of polyketide and peptide biosynthesis (Takita and Muroka (1990) pages 289-309 In: Biochemistry of Peptide Antibiotics: Recent Advances in the Biotechnology of β-Lactams and Microbial Peptides, Kleinkauf and Von Döhren Eds., W. de Gruyter, New York). On the assumption that BLM biosynthesis follows the paradigm for peptide and polyketide biosynthesis, we predict that the Blm megasynthetase, which catalyzes the assembly of the BLM backbone from nine amino acids and one acetate, should bear the characteristics of both NRPS and PKS, providing an excellent model to study the mechanism by which NRPS and PKS could be integrated into a productive biosynthetic system to synthesize a hybrid peptide and polyketide metabolite (FIG. 1A) (Shen et al. (1999) Bioorg. Chem. 27: 155).